1. Field of the Invention
This invention pertains to the disruption of bacterial biofilms with antibacterial enzymes. More specifically, this invention relates to the disruption of staphylococcal biofilms with lysostaphin.
2. Background Art
A. Biofilms
Bacteria that adhere to implanted medical devices or damaged tissue can encase themselves in a hydrated matrix of polysaccharide and protein and form a slime layer also known as a biofilm. Biofilms pose a serious problem for public health because of the increased resistance of biofilm-associated organisms to antimicrobial agents and the association of infections with these organisms in patients with indwelling medical devices or damaged tissue. Antibiotic resistance of bacteria growing in biofilms contributes to the persistence and chronic nature of infections such as those associated with implanted medical devices. The mechanisms of resistance in biofilms are different from the now familiar plasmids, transposons, and mutations that confer innate resistance to individual bacterial cells. In biofilms, resistance seems to depend on multicellular strategies.
Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces or damaged tissue. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces as biofilms. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities.
The biofilm matrix is a dynamic environment in which the component microbial cells appear to reach homeostasis and are optimally organized to make use of all available nutrients. The matrix therefore shows great microheterogeneity, within which numerous microenvironments can exist. Biofilm formation is believed to be a two-step process in which the attachment of bacterial cells to a surface is followed by growth dependent accumulation of bacteria in multilayered cell clusters. Although exopolysaccharides provide the matrix framework, a wide range of enzyme activities can be found within the biofilm, some of which greatly affect structural integrity and stability.
More specifically, during the first phase of formation, it is hypothesized that the fibrinogen and fibronectin of host plasma cover the surface of a medical implant or damaged tissue and are identified by constitutively expressed microbial surface components, which mediate the initial attachment of bacteria to the surface of the biomaterial or damaged tissue. In the second step, a specific gene locus in the bacteria cells, called the intracellular adhesion (ica) locus, activates the adhesion of bacteria cells to each other, forming the secondary layers of the biofilm. The ica locus is responsible for the expression of the capsular polysaccharide operon, which in turn activates polysaccharide intercellular adhesion (PIA), via the sugar poly-N-succinylglucosamine (PNSG), a-1,6-linked glucosaminoglycan. The production of this polysaccharide layer gives the biofilm its slimy appearance when viewed using electron microscopy.
Staphylococcus aureus is a highly virulent human pathogen. Both S. aureus and coagulase-negative staphylococci have emerged as major nosocomial pathogens associated with biofilm formation on implanted medical devices and damaged tissue. These organisms are among the normal carriage flora of human skin and mucous membranes, making them prevalent complications during and after invasive surgery or prolonged hospital stays. As bacteria carried on both healthy and sick people, staphylococci are considered opportunistic pathogens that invade patients via open wounds and via biomaterial implants.
Biofilm infections associated with S. aureus are a significant cause of morbidity and mortality, particularly in settings such as hospitals, nursing homes and infirmaries. Patients at risk include infants, the elderly, the immuno-compromised, the immuno-suppressed, and those with chronic conditions requiring frequent hospital stays. Patients with intravascular and other implanted prosthetic devices are at even greater risk from staphylococcal infections because of compromised immune systems and the introduction of foreign bodies, which serve to damage tissue and/or act as a surface for the formation of biofilms. Such infections can have chronic, if not fatal, implications.
Catheter related infections continue to be a significant source of morbidity and mortality in patients requiring catheterization. The reported incidence in the United States is 4%, which equates to 200,000 patients per year. Additionally, catheter related infections have an attributable mortality of 14-24% and increase medical expenses by prolonging hospitalization. As a result, prevention or even reduction in the incidence of these catheter-related infections could have a significant healthcare benefit.
Catheter infections are most commonly caused by staphylococci, either coagulase negative staphylococci (CoNS) or S. aureus. Infections caused by CoNS can be mild and some can be treated by either removing the catheter or a course of antibiotics with the catheter in place. S. aureus infections are usually more severe and require removal of the catheter or other prosthetic device in addition to extended antibiotic therapy.
S. aureus is a prodigious toxin producer and a highly virulent human pathogen. It is the cause of a variety of human diseases, ranging from localized skin infections to life-threatening bacteremia and infections of vital organs. If not rapidly controlled, a S. aureus infection can spread quickly from the initial site of infection to other organs. Although the foci of infection may not be obvious, organs particularly susceptible to infection include the heart valves, kidneys, lungs, bones, meninges and the skin of burn patients.
While effective antimicrobial agents against antibiotic-susceptible staphylococcal infections have been developed, agents are still needed that consistently and thoroughly kill antibiotic-resistant S. aureus especially those associated with biofilms, on implanted prosthetic devices and on damaged tissue, to eliminate this source of persistent and chronic staphylococcal infections. Unfortunately, S. aureus in biofilms (even those which are antibiotic-susceptible in the planktonic state) tend to be less susceptible to antibiotics and thus a more difficult infection to clear.
The causes of biofilm resistance to antibiotics may include, the failure of some antimicrobial agents to penetrate all the layers of a biofilm, the slow-growth rate of certain biofilm cells that make them less susceptible to antimicrobial agents requiring active bacterial growth, and the expression of gene patterns by the bacterial cells embedded in the biofilm that differ from the genes expressed in their planktonic (free-swimming) state. These differences in biofilm-associated bacteria render antimicrobial agents that work effectively to kill planktonic bacteria ineffective in killing biofilm-associated bacteria. Often the only way to treat catheters or prosthetic devices with associated biofilms is the removal of the contaminated device, which may require additional surgery and present further risks to patients.
Coating catheters on other prosthetic devices with anti-microbial agents is a promising approach for the control and prevention of these foreign body related infections. Currently, six types of antiseptic catheters have been tested in clinical trials: cefazolin, teicoplanin, vancomycin, silver, chlorohexidine-silver sulfadiazine and minocycline-rifampin coated catheters. However, only the minocycline-rifampin coated catheters have been shown to reduce the incidence of catheter related bloodstream infections (CRBI's), and its long-term efficacy has not been investigated. There is a clear need to find a new antimicrobial agent with properties that improve catheter durability by decreasing CRBI's and an agent that has the capacity to clear biofilm associated staphylococcal infections in place, be they on catheters, prosthetic devices or damaged tissue, without requiring surgical removal.
B. Lysostaphin
One such anti-microbial agent that was originally believed to be ineffective against biofilms is lysostaphin. Lysostaphin is a potent antibacterial enzyme first identified in Staphylococcus simulans (formerly known as S. staphylolyticus). A bacterial glycylglycine endopeptidase, lysostaphin is capable of cleaving the specific cross-linking polyglycine bridges in the cell walls of staphylococci, and is therefore highly lethal to both actively growing and quiescent staphylococci. Expressed in a single polypeptide chain, lysostaphin has a molecular weight of approximately 27 kDa.
Lysostaphin is particularly effective in lysing S. aureus because the cell wall bridges of S. aureus contain a high proportion of glycine. Lysostaphin has also demonstrated the ability to lyse Staphylococcus epidermidis, the most prevalent coagulase-negative bacterial infection found in hospital settings. However, because of the complexity of biofilm architecture and the mechanism by which lysostaphin lyses staphylococci, lysostaphin was not expected to be effective against staphylococci in established biofilms.
U.S. Pat. No. 6,028,051 to Climo, et al., discloses a method for the treatment of staphylococcal disease with lysostaphin. Relatively high doses of lysostaphin, of at least 50, preferably 100, milligrams of lysostaphin per kilogram of body weight are used for treatment. Lysostaphin can be used in single dose treatments or multiple dose treatments, as well as singularly or in combination with additional antibiotic agents. The '051 patent also discloses that the cloning and sequencing of the lysostaphin gene permits the isolation of variant forms that can have properties similar to or different from those of wild type lysostaphin.
U.S. Pat. No. 6,315,996 to O'Callaghan, discloses a method for using lysostaphin as an effective antibiotic for topical treatment of staphylococcus corneal infections. U.S. Pat. No. 5,760,026 to Blackburn et al., discloses a method for using lysostaphin to eliminate and cure staphylococcal infections including the cure of mastitis in dairy cows by intramammary infusion.
U.S. Published Patent Application No. 2002/0006406 filed by Goldstein et al. discloses that low doses of lysostaphin, on the order of 0.5 to 45 mg/kg/day, and its analogues such as variants and related enzymes, are “sufficient” to eradicate most staphylococcal infections, including those “associated with” a catheter or prosthetic device. Thus, there is no disclosure in this or the other publications to lead one skilled in the art to expect lysostaphin to be effective for disrupting biofilms of staphylococcal or other bacterial origin established on the surface of implanted prosthetic devices, catheters or damaged tissue. It should be noted that, not all bacteria “associated with” a catheter or prosthetic device are in biofilms, and not all biofilms are “associated with” catheters or prosthetic devices.